Project Summary HIV-1 capsid protein (CA) determines the virus nuclear entry and integration site preference. By developing novel tools to track single viral complexes that establish infection, we have recently found that point mutations in CA (N74D) influence the targeting of viral integration to the periphery as opposed to interior of the nucleus preferred by wild-type virus. A nuclear role for CA, which remains poorly appreciated, is potentially derived from the subset of CA molecules that remain associated with nuclear pre-integration complexes (PICs). We hypothesize that interaction between PIC-associated CA molecules and cellular co-factor CPSF6 directs the transport of HIV-1 to nuclear speckle regions that are rich in actively transcribing genes for integration. The scientific premise of this proposal is to characterize the CA/CPSF6 dependent nuclear HIV-1 transport to the sites of integration. We will, (1) apply live-cell imaging in combination with photoactivation techniques to visualize CPSF6 interaction with fluorescent CA-labeled PICs and their transport to sites of integration. (2) Determine a role for CPSF6/PICs interaction in the HIV-1 nuclear transport by determining diffusion coefficients of single particles in the presence of drugs and CA mutants that abrogate CA/CPSF6 interactions. (3) Develop a live-cell imaging assay to visualize the integrated vDNA, and correlate the location of PICs disappearance to integration. (4) Determine the amino acid residues in CA involved in its interaction with viral RNA or proteins in PICs and identify the binding partner of CA in vRNPs. This new direction of research will delineate a role for CA in interactions and intra-nuclear trafficking of PICs to locations of HIV-1 integration, an important step in viral life cycle that remains poorly appreciated.